Only a few RGCs survive after ONC and even fewer can extend their axons through the injury site,
51 with different subtypes showing dramatic differences in the survival rate and ability to regenerate their axons.
16 αRGCs have been previously shown to survive better than many other RGC types and account for nearly all the regeneration seen following downregulation of PTEN and subsequent activation of the mTOR pathway.
16 Furthermore, αRGCs preferentially express OPN and receptors for insulin-like growth factor 1. Overexpression of these proteins promotes regeneration of αRGC axons.
16 Other factors and signaling pathways might be also involved in RGC neuroprotection and stimulation of axon regeneration.
10 One of these factors is the transcription factor ATF3, known to be inducible by stress and axonal injury in both the peripheral nervous system and CNS.
27,30 In the adult intact retina, expression of ATF3 is not detected in RGCs; however, a fraction of RGCs were shown to upregulate ATF3 after ONC.
22 In this paper, we demonstrated that more than 80% of αRGCs surviving after ONC were positive for ATF3 expression while only 30% of non-αRGCs were ATF3
+ (
Fig. 1D). Although we did not analyze the nature of surviving ATF3
+ non-αRGCs, we assumed that some of these cells could be melanopsin expressing M1-RGCs because these cells, similar to αRGCs, preferentially survive after ONC.
16 Indeed, it has been recently demonstrated that, similar to αRGCs, the level of
Atf3 gene expression is very low in melanopsin expressing RGCs in a control undamaged mouse retina and is dramatically upregulated in these cells after ONC.
52 We showed that overexpression of ATF3 by intravitreal injection of rAAV-ATF3 before ONC promoted survival of RGCs for at least 2 weeks (
Fig. 2). The neuroprotective effect of ATF3 overexpression was similar to that of neuritin, a known RGC neuroprotective factor.
7,53 However, a single rAAV-ATF3 injection was not sufficient for RGC neuroprotection over 6 weeks. This indicates that ATF3 effects on injured RGCs may be transient or that multiple rAAV-ATF3 injections are needed, similar to a rat glaucoma model where RGC neuroprotection by intravitreal injections of small extracellular vesicles required monthly injections while a single injection was not effective over 2 months.
54 Stimulation of RGC axon regeneration by ATF3 after ONC was minor with no statistically significant differences between control and ATF3 samples detected (
Fig. 3). Although abilities of a particular RGC type to survive and regenerate axons after ONC very often correlate, this is not always the case. For example, melanopsin-expressing M1-RGCs and αRGCs survive preferentially after ONC but only the αRGCs, not M1-RGCs, regenerate their axons.
16 Overexpression of Sox11 kills αRGCs yet promotes the regeneration of non-αRGC axons after ONC.
25 It appears that overexpression of ATF3 is not sufficient to activate a set of genes essential for RGC axon growth.